Cosmetic Preparation and Method for Preparing the Same

ABSTRACT

The present invention relates to the use of dedifferentiated plant cells in cosmetic preparations for protecting of stem cells against intrinsic and extrinsic stress factors, in particular for promoting proliferation of stem cells and for protecting them against apoptosis. In particular, the invention relates to the use of dedifferentiated plant cells from fruits of  Malus domestica  (Apple) cultivar  Uttwiler Spaetlauber . Further, the invention relates to a method for cultivating of dedifferentiated plant cells, as well as to the preparation of extracts of plant cell cultures which are suitable for such applications.

REFERENCE TO RELATED APPLICATION

The present application is a continuation of and claims priority toco-pending application Ser. No. 12/148,241, which was filed on Apr. 17,2008, the entire disclosure of which is incorporated herein byreference.

FIELD OF INVENTION

The present invention relates to the use of dedifferentiated plant cellsin cosmetic preparations for protecting of stem cells against intrinsicand extrinsic stress factors, in particular for promoting proliferationof stem cells and for protecting them against apoptosis. In particular,the invention relates to the use of dedifferentiated plant cells fromfruits of Malus domestica (Apple) cultivar Uttwiler Spaetlauber.Further, the invention relates to a method for cultivating ofdedifferentiated plant cells, as well as to the preparation of extractsof plant cell cultures which are suitable for such applications.

BACKGROUND OF THE INVENTION

Stem cells (SC) are uniform undifferentiated cells having the propertyof constant regeneration and the unique ability of turning into anyother cell type by cleavage and differentiation. By said potential, SCare a renewable source of human tissue. Thus, SC be-came an importantobject of medical research for various applications, such as genetherapy, organ transplantation, diabetes, and plastic surgery.

SC may be divided into two groups, i.e. embryonic and adult (EC).Embryonic stem cells play a key role in the first development phase ofan organism. They are able to endlessly cleave and to develop everynecessary type of tissue. Thus, they are able to form from a single cella whole body, either the plant or the animal form which they originate.Due to this ability they are also called pluripotent cells.Unfortunately, for human beings this ability is restricted to theembryonal phase. In later phases of life of a subject EC are no longerpresent.

The second type of stem cells are adult stem cells. So far, these cellscould be identified in many full-grown tissues and organs, such as bonemarrow, pancreas, spine, brain, central nervous system, peripheralblood, dental pulp, blood vessel, skeletal muscles, cornea, retina,liver, cord blood, heart, epithelium of the intestinal tract, anddermis.

Compared with EC adult SC derived from such tissues have only a limitedchoice of differentiation. Some of them can only differentiate into onesingle tissue type mostly the one which is surrounding them. Thus, theyare called unipotent SC. Other SC can differentiate into various tissuetypes and therefore are called multipotent.

Both types of adult SC are promising for medical applications since theyare more easily accessible and their recovery is ethically lessproblematical than of ES. A survey on SC and their possibilities may begleaned in Lemoni et al., 2005, Stem Cell Plasticity: Time for aReappraisal, Hematologica/The Hematology Journal, 90 (3), 360 to 381.

The skin of mammals is a multilamina system which is continuouslyrevolving. The part which is constantly in contact with the outsideworld is called epidermis. The major task of this specialized Tissue isto protect the body against dehydration, lesions and infections. It iscomposed four different laminas which are all formed by a single celltype, the so-called cerationocytes. Whereas this cell type is not muchdifferentiated, is nevertheless has its origin in specialized skin stemcells. They are located in the lowermost lamina of the epidermis, thebasal lamina.

In several papers successful isolation of such skin stem cells isreported. It even could be demonstrated that skin stem cells may befound in lower tissues of the skin, i.e. in the so-called hair folliclebulge. Contrary to the SC in the basal lamina these SC are multipotent,i.e. they are able to differentiate into every tissue type of the skin.A survey may be gleaned in: Roh et al., 2006, Cutaneous Stem Cells andWound Healing, Pediatric Research, 59 (4), Pt 2, 100 to 103R; Morasso etal., 2005, Epidermal Stem Cells; The Cradle of Epidermal Determination,Differentiation and Wound Healing, Biol. Cell., 97, 173 to 183; andAlonso et al., 2003, Colloquium: Stem Cells of the Skin Epithelium,PNAS, 100, Suppl. 1, 11830 to 11835.

Skin stem cells are crucial in the wound healing and the regeneration ofskin and hair. However, the capacity of these abilities may be disturbedby genetic problems, environmental influences and the aging process.Thus, protection of these SC is extremely important. Therefore, as willbe explained below more in detail, it was an object of the presentinvention to develop a plant extract able to protect and stimulate theseSC in cosmetic preparations.

Plant extracts and the use of parts of plants, such as leaves, fruits,flowers, stems, bark, inflorescences and roots for cosmetic and medicalApplications are known since ancient times. Products derived therefrommay be, e.g., essential oils, fibers, starch, flavors, coloring matters,antibiotics, proteins, phenols, acids or fats. The use of plants orplant extracts in cosmetics is rampant. There are a great many ofdifferent uses, such as humidification, brighteners, tanning lotions,make-ups, sun filters, scavengers, antioxidants, immunity stimulation,detergents, preserving agents or thickening agents.

Examples of recently found uses are described in: KR20040091178,KR20040059007, US20062400129, WO2006099930, WO2006086707, US2006153792,JP2006151934, WO2006068777, WO2006053761, WO2006008418, LV13345,UA73556, CN1679498, and many others.

The spectrum of useful plants and plant component is wide and comprisese.g. algae, succulents, berries, carnivorous plants, herbs, cereals andtrees. Usual well known examples of plants, however not limited to them,are: Spirulina algae, aloe vera,

calendula, ginkgo, ginseng, iris, valerian, sage, lavender, thyme,peppermint, Saint-John's-wort, citrons, peach, guava, avocado, wheat,and oat.

However there are restrictions with respect to the use of plants orplant components. i.e.:

-   -   The availability may be restricted, e.g. by the seasons, limited        storage capacities, protection of species, problems in        cultivation, or bad harvests.    -   The quality is not unchanging, e.g. due to seasonal variations,        different cultivation methods, geographic differences, different        suppliers, clones, pollution of the environment, or physical        status.

These facts often make the use of plants in cosmetic applicationsimpossible.

Therefore, the utilization of methods of plant cell culture techniquesmay help to solve such problems. Said utilization comprises techniqueswhich allow, when observing certain known process steps, to obtainuniform dedifferentiated cells, showing the following advantages ascompared with cultivated whole plants:

-   -   Independency from seasons;    -   Continuous production:    -   Freedom from pollution of environment and other impurities;    -   With respect to quantity and quality manageable and reproducible        production of metabolites;    -   Protection of rare or limited plant reserves;    -   No limitation of market availability.

Examples for the use of plant cell cultures of various species, theircultivation and their use in cosmetic preparations may e.g. be found in:EP1244464, FR2837385, US2006021084, WO2005108596, WO2005070066.

The basic principle of cultivation of such dedifferentiated plant cellsutilizes the biological fact, that every plant cell has the ability tobuild up the whole plant which the cell stems from. This ability iscalled totipotency and is comparable with the pluripotency of animal ES.Therefore, it may be accepted that dedifferentiated plant cells do havea positive influence on protection and activation of skin stem cells.

In order to achieve this effect various dedifferentiated plant cells canbe used. However, further supplemental useful effects can be achievedusing these plant extract as well. An investigation within several plantgroups showed that apples and fruits belonging to the subfamilyMaloideae of the family of Rosaceae are much promising. A known exponentof this family is the cultivated apple tree (Malus domestica). Appleshave a long tradition in cosmetic applications. Originally, they wereapplied in the form of masks of pressed pulp or peelings which providedmoisture and tautness of the skin. Another application is the use ofapple aromas and extracts in all kinds of cosmetic preparation, such ase.g. shampoos, lotions, soaps, bath essences or toothpastes.

The main ingredients of apples are various sugars, vitamins, acids,oils, waxes and polyphenols. Recently published studies could prove thatespecially the overall polyphenols in apple extracts or apple juicesrich in polythenols can be useful in preventing and combating coloncancer (Eberhart et al., 2000, Antioxidant Activity of Fresh Apples,Nature, 405, 903 to 904; Liu et al., 2003, Antiproliferative Activity ofApples is not due to Phenolic-induced Hydrogen Peroxide Formation, J.Agric. Food Chem., 51, 1718 to 1723; Kern et al., 2005, Inhibitors ofthe Epidermal Growth Factor Receptor in Apple Juice Extract, Mol. Nutr.Food Res., 49, 317 to 328). Thereby, to a certain extent, the juicesrich in polyphenols had an influence on the Wnt-pathway. This pathway isa cytobiological signaling cascade in which p-catenin is the mainprotein. Under normal circumstances, this protein is present in the cellon a constant level. If this level is disturbed, as in a cancer cell,the p-catenin level rises, and the p-catenin is transported into thecell nucleus, where it initiates the transcription of genes which causesan uncontrolled cleavage. Kern et al. (2006, Modulation of Key Elementsof the Wnt-Pathway by Apple Polyphenols, J. Agric. Food. Chem., 54, 7041to 7046) could show that the level of intracellular p-catenin in coloncancer cells cultivated in vitro was reduced by administration of applejuice.

Furthermore, it was found that apple show a large antioxidative activityand can increase the antioxidative capacity in blood (Rezk et al., 2002,The Antioxidant Activity of Phloretin: The Disclosure of a newAntioxidant Pharmacophore in Flavonoids, Biochem. Biophys. Res. Commun.,295, 9 to 13; Lee et al., 2003, Major Phenolics in Apple and theirContribution to the Total Antioxidant Capacity, J. Agric. Food Chem.,51, 6516 to 6520; Vrohovsek et al., 2004, Quantitation of Polyphenols inDifferent Apple Varieties, J. Agric. Food Chem., 52, 6532 to 6538;Lotito et al., 2004, Relevance of Apple Polyphenols as Antioxidants inHuman Plasma: Contrasting in-vitro and in-vivo Effects, Free Rad. Biol.Med., 36, 201 to 211; Bitsch et al., 2000, Bioavailability ofAntioxidative Compounds from Brettacher Apple Juice in Humans, Food Sci.Emerg. Technol., 1, 245 to 249). For this reason, apples are veryinteresting for establishing a dedifferentiated cell culture and itssubsequent use in cosmetic preparations.

Dedifferentiated plant cells have a complex matrix of constituents ofsalts, acids, polyphenols, sugars, fats, proteins and other components.In addition to known components there is an unknown fraction ofcomponents which possibly is very valuable for cosmetic applications. Itis known that raw plant extracts often show a better effect thanidentified and isolated individual components. Therefore, it isreasonable to use the entire cell lysate for application.

In order to obtain such total fraction of all ingredients specialtechniques are required since part of them are water-soluble whereasanother part is fat-soluble. It was pro-posed to process plant cellculture preparations by means of lyophilization (e.g. WO2005072697,US20050265953). Thereafter, these lyophilized cells were pulverized andused in topic preparations.

Since transport of materials through the skin barrier is very limited,the technique of producing liposomes for many cosmetic applications wasdeveloped (e.g. KR20050091162, KR920005639B, GB2415375, WO2004067012,EP1498420, US2002160064, AU2388099). Application of this techniqueallows a better penetration of substances into the lower skin laminas.Also, a further advantage of liposomes is the encapsulation offat-soluble ingredients in the membrane and thus their dispersion inaqueous phases.

There are various methods of liposome production. Main steps of theirproduction comprise dissolving a phospholipid mixture in a suitablesolvent (e.g. glycerol or alcohol), intermixing the dissolved lipidswith an aqueous phase, applying energy (e.g. by stirring, shaking,pressure or heat) for forming the liposomes. As said above, the form ofenergy can by pressure. Formation of liposomes by means of high pressurehomogenization is a known technique. Examples for pharmaceutical orcosmetic preparations may be found e.g. in WO9949716, NZ502840 orEP0782847. Interestingly the same technique can be used for solubilizingcells and obtaining their lysate (e.g. DE19918619). Therefore, it ispossible to solubilize plant cells of suspension cultures and at thesame time to extract the oil- and water-soluble agents into emptyliposomes. Thereby, stability of the agents and their transportationinto the skin can be improved.

From the above mentioned publication WO 2005/072697 A1 it is known touse lyophilizates of dedifferentiated plant cells for depigmenting theskin. This technique calls for the use of lyophilizates ofdedifferentiated plant cells, in particular of cells of halophileplants. A use for stimulation and protection of skin stem cells is notenvisaged.

From the publication EP 1,174,120 A1 it is known to use extracts, inparticular of lyophilizates, of dedifferentiated cells of plants of thefamily Iridaceae (Iris-family) for stimulating immunity. Other plants oruses are not proposed.

The publication EP 1,064,932 A1 proposes the use of extracts ofdedifferentiated plant cells in deodorants. Other uses are notdisclosed.

The publication WO 03/077881 A discloses the use of lysates ofmetabolites of dedifferentiated cells of vine, which were obtained bymeans of a complicated method, for the preparation of cosmetics. Thistechnique calls for the use of lyophilizates. A use for stimulation andprotection of skin stem cells is not envisaged. Furthermore, otherspecies of plants are not disclosed.

Furthermore, the publication WO 01/47538 A1 discloses the use ofextracts of dedifferentiated cells of plants of the genus Leontopodium(Edelweiss) as UV filter. Other uses or other plants are not proposed.

OBJECTS OF THE INVENTION

The main object of the present invention is to create a cosmeticpreparation which protects stem cells against intrinsic and extrinsicstress factors, in particular promotes the proliferation of stem cellsand protects them against apoptosis.

Another object of the present invention is to provide a method forpreparing an extract suitable for use in said cosmetic preparation.

SUMMARY OF THE INVENTION

The abovementioned object is achieved by using an extract of asuspension of dedifferentiated plant cells, preferably of the family ofRosaceae (Rose family), particularly of the subfamily Maloidae (Pomefruit), and more in particular of Malus domestica cultivar UttwilerSpaetlauber, which is an old and rare kind of apple.

The method of preparing suitable extracts comprises the following mainsteps:

-   -   (a) Establishing a stable dedifferentiated cell line on a        laboratory scale;    -   (b) Mass cultivation of the cells in an one-way bag reactor        system (so-called Wave reactor), and    -   (c) Recovery of a total-extract by means of high pressure        homogenization using empty liposomes.

Advantageously, in step (c) the following procedure is followed:

-   -   Decomposition of the plant cells by high pressure        homogenization;    -   Extraction and stabilization of the substances of content by        means of liposomes;

whereby both steps are simultaneously performed as one single step.

BRIEF DESCRIPTION OF DRAWINGS

In the annexed drawings:

FIG. 1 shows the increase in the cell count of umbilical cord stem cellsin dependence of different concentrations of a liposomal extractoriginating from dedifferentiated cells of apples of the cultivarUttwiler Spaetlauber. For this study, the extract was centrifuged andsterilized by filtration.

FIG. 2 shows the increase in proliferation capability in a MTS-assay ofumbilical cord stem cells in dependence of different concentrations of aliposomal extract originating from dedifferentiated cells of apples ofthe cultivar Uttwiler Spaetlauber. For this study, the extract wascentrifuged and sterilized by filtration.

FIG. 3 shows microscopic pictures of umbilical cord stem cells. The leftphotograph shows cells cultivated in a medium without extract, the rightphotograph cells cultivated together with 0.1 percent of a liposomalextract originating from dedifferentiated cells of apples of thecultivar Uttwiler Spaetlauber. For this study, the extract wascentrifuged and sterilized by filtration.

FIG. 4 shows the proliferation capability of umbilical cord stem cellsof a control preparation and of a liposomal extract of differentconcentrations originating from dedifferentiated cells of apples of thecultivar Uttwiler Spaetlauber, 48 hours after UV irradiation. For thisstudy, the extract was centrifuged and sterilized by filtration.

FIG. 5 shows the temporal influence of a liposomal extract according toExample 10 on the length of hair follicles.

FIG. 6 shows the effect of a preparation in accordance with the presentinvention as anti-wrinkle cream in Test 1 described hereafter.

FIG. 7 shows the effect of a preparation in accordance with the presentinvention on stressed skin in Test 2 described hereafter.

In FIGS. 2 and 4 “OD” is the abbreviation of “optical density”.

DETAILED DESCRIPTION OF THE INVENTION Induction and Stabilization ofCell Line (a)

The following steps provide a dedifferentiated cell line from planttissue:

-   -   (a1) Choice of a suitable tissue for the induction.    -   (a2) Surface sterilization.    -   (a3) Cladding of the explanates on a suitable solid medium for        callus induction.    -   (a4) Harvesting the callus grown on the injured surface of the        explanates.    -   (a5) Sub-cultivation of the obtained callus on the same medium        until the cells are entirely dedifferentiated.    -   (a6) Addition of the dedifferentiated cells to a suitable liquid        medium.    -   (a7) Homogenization of the cells in suspension until big cell        clump are no longer present; and    -   (a8) Sub-cultivation and continuous characterization of the cell        suspension.

Fundamental working protocols for plant cell cultures can be found inthe standard literature (e.g. Plant Cell Culture: A Practical Approach,Editor P. A. Dixon, 1994, Oxford University Press). Protocols for thework and suitable media for initiating plant cell cultures of apples aredescribed by Nitsch et al., 1970, Bases physiologiques de la productionde chair de pomme et de poire in vitro, Bull. Soc. Bot. Fr., 117, 479 to492; and Pech et al., 1975, Croissance in vitro de tissues et desuspensions cellulaires de pomme, Bull. Soc. Bot. Fr., 122, 183 to 194.According to these protocols, initiating and maintaining such culturesshould not present a problem.

Biomass Production (b)

In the following process, the obtained suspension culture is cultivatedfurther over several continuous steps from small laboratory flasks(Erlenmeyer flask usually having 200 ml content) to production scale of50 to 100 liters. In this process, 5 to 10 percent, preferably 10percent, of the next culture volume of a fully grown cell suspension isused as inoculum. The scale-up may be done in steps of e.g. 0.1/1/10/100liter.

Cultivation volumes exceeding 1 liter necessitate the use of specialbioreactors instead of culture flasks used before. Many differentsystems are available on the market. Execution of cultivation is done,but is not limited thereto, in agitation reactors, bubble columns, loopreactors or newly developed one-way systems suitable for plant cellcultivation. For all these cultures the influence of shearing stresswhich can damage the cultures. Thus, the most important parameter forselecting a suitable reactor system usually is the manner how theculture is homogenized.

Moreover, control of the culture is very important. In comparison tocultures of yeast or bacteria, measurement of the biomass is difficult,and the growth of biomass has to be measured by means of indirectparameters, such as e.g. consumption of carbon, dropping of conductivityor the pH value or the increase of optical density. Once such a controlis established, the end point or the harvest moment, respectively, canbe fixed.

Also important is examination analysis of secondary metabolites whichare characteristic for the cell culture. Measurement of such materialscan be done by HPLC-VIS/UV/MC, LC, GC-MS, e.g. enzymatically oroptically. Thereby, the stable and continuous expression of suchmetabolites during the whole process is decisive.

Biomass Processing (c)

In order to obtain an extract containing the whole essence of thecultivated cells, the cells are solubilized by means of liposomes. Themain component of this method is the use of high pressure homogenizationof the whole cell broth together with a liposome preparation. The greatadvantage of this method is its simple and low-cost application.

In detail, the method comprises the following steps:

(c1) Addition of a suitable liposome preparation to the cell broth;

(c2) Addition of a suitable preservative agent;

(c3) Addition of suitable antioxidants;

(c4) Mixing the substances; and

(c5) High pressure homogenization.

All preservative agents of natural or synthetic origin allowed forcosmetics, such as e.g. phenoxyethanol, benzoic acid, propionic acid,alcohol or silver chloride, can be used as preservative agents.

In order to additionally protect the extract from oxidation,antioxidants, such as e.g. ascorbic acid or tocopherol, may be added.

The described method allows the addition of still further substancesuseful in the preparation or cosmetic product. Once all compounds areadded, the mixture has to be stirred in order to dissolve thepreservative agents and other components. This may be done e.g. by meansof a paddle mixer, a homogenization rod or by pumping through staticmixing elements.

The subsequent high pressure homogenization pursues to objects:

-   -   Destruction of the cell membranes in order to release        extractable substances; and    -   Generation of finely dispersed liposomes contained the fat- and        water-soluble fractions of the cells.

Suitable high pressure homogenizators are commercially available on themarket. The principle of the reaction chamber has to be selected fromdifferent possibilities and has to be previously tested. The number ofpassages through the reaction chamber necessary for a disintegration ofall cell membranes or reaching a desired homogeneity of the extract hasto be tested as well.

Afterwards, the extract obtained in this manner can directly beincorporated into cosmetic preparations, such as e.g. creams, soaps,lotions, gels or hair seras. If the extract is to be used assemi-finished good a supplemental thickening is possible. All thickeningagents of natural or synthetic origin allowed for cosmetics can be usedas thickening agents.

EXAMPLES Example 1 Production of a Dedifferentiated Plant Cell Culture

Mature apples of the cultivar Uttwiler Spaetlauber were rinsed with tapwater. In cylindrical pieces of a diameter of about one centimeterdiameter the score was out along the axis of the by means of a corkborer. For the surface sterilization, the cylinders were dipped for 30seconds into 70 percent ethanol and thereafter for 10 minutes into 2.5percent sodium hypochloride containing 0.1 percent of the surfactantTween 40. Thereafter, the sterilized cylinders washed tree times withdistilled water, cut into slices of about 3 millimeter thickness, andplaced on a solid medium of the following composition per liter:

Calcium chloride 332 mg Potassium dihydrogen phosphate 170 mg Potassiumnitrate 1900 mg Magnesium sulfate 180.54 mg Ammonium nitrate 1650 mgCobalt chloride hexahydrate 0.025 mg Copper sulfate pentahydrate 0.025mg Iron-Sodium-EDTA 36.7 mg Boric acid 6.2 mg Potassium iodide 83 mgManganese sulfate hydrate 16.9 mg Disodium molybdate dihydrate 0.25 mgZinc sulfate heptahydrate 8.6 mg myo-Inositol 100 mg Nicotinic acid 5 mgGlycine 2 mg Pyridoxine hydrochloride 0.5 mg Thiamidine hydrochloride0.5 mg Folic acid 0.5 mg Biotin 0.05 mg Ascorbic acid 50 mg Thiourea 25mg L-Asparagine 180 mg Saccharose 30000 mg

The ph-value was adjusted to 5.6 with sodium hydroxide solution. Agarwas added in a concentration of 0.8 percent as gelling agent. Allingredients were mixed together and sterilized at 121° centigrade for 15minutes.

The induction of the primary callus was carried out in the dark at 25°centigrade. The formed calluses were harvested after two to three weeksand further incubated on the same medium. Several sub-cultivations werecarried out until the callus was fully dedifferentiated.

Example 2 Production of a Suspension Culture

Dedifferentiated cell clumps growing on said solid medium were taken,homogenized and placed into the same medium without gelling agent. Afinely dispersed suspension was obtained which could be use for largercultivation systems. The suspensions were grown in the dark at 25centigrade and a shaking velocity of about 100 rpm.

Example 3 Outbreak

One tenth of a fully grown culture (percentage of cells being about 50percent of the total weight of the culture) was used for the seeding ofthe next volume step. The scale-up was effected in a one-way bag reactorsystem of Wave Biotech AG, Tagelswangen, Switzerland (so-called WaveReactor). The scale-up was effected in steps of 1/10/25 liter. Thetemperature was held at 25 centigrade and the aeration at about o.1 vvm.Various mixing speeds were applied in dependence of the bags used.Cultivation was carried out in the dark, and it took about 20 days untila bag was completely grownup.

Example 4 Preparation of a Liposomal Extract

After cultivation, the whole cell broth was mixed with a dispersioncontaining empty liposomes of a size of about 50 nanometer. The mixturewas then four times high pressure homogenized at a pressure of about1200 bar (1.2*10⁸ N m⁻²) resulting in a finely dispersed extract.

Example 5 Vanishing Cream

The percentage refers to the total quantity (weight/weight).

Oily phase 1: Alkyl benzoates 10%  Dimeticone 3% Archidyl glycosides 3%Myristyl glycoside 2% Oily phase 2: Polyacrylamides 1% Aqueous phase:Demineralized water 71%  Glycerol 5% Phenoxyethanol 1%

Oily phase 1 and the aqueous phase were heated at 80 centigrade andblended. The mixture was chilled to 60 centigrade. Then oily phase 2 wasadded, and the mixture was blended. The mixture was chilled to 30centigrade. 4 percent of the extract described in Example 4 was addedand the mixture was blended again.

Example 6 Liquid Balm for the Scalp

The percentage refers to the total quantity (weight/weight).

Ethanol 0.5% Urea   5% Propylene glycol 0.5% Carbomer 0.4% Bisabololene0.1% PEG-60 0.6% D-Panthenol 75% 0.5% Sodium hydroxide 30% 0.4% Plantcell extract of Example 4   1% Water filling up to 100%

Example 7 Intensive Hair Mask

The percentage refers to the total quantity (weight/weight).

Phase Ingredient Amount Aqueous phase 1 (W1) Water filling up to 100%Citric acid 0.6% Sodium benzoate 0.5% Aqueous phase 2 (W2) D-Panthenol75% 0.7% Oily phase 1 (O1) Cetearyl alcohol 4.5% Dicocoylethyl   3%hydroxyethlmonium methosulfate Distearoylethyl 1.5% hydroxetylmoniummethosulfate Dicapryryl ether   1% Gycerol stearate   1% Oily phase 2(O2) Amino dimethicone 0.3% Plant extract (A) Pant cell extract   2% ofExample 4

Instruction for Preparation:

Aqueous phase 1 is mixed and heated to 75 centigrade. Shortly beforemixing with oily phase 2 aqueous phase 2 (panthenol) is added. Oilyphase 1 is heated to 75 centigrade, and shortly before mixing oily phase2 (aminodimethicone) is added. The combined aqueous and oily phases aremixed and homogenized. The mixture is chilled to 30 centigrade, andphase A (plant extract) is added.

Example 8 Eye Cream

The percentage refers to the total quantity (weight/weight).

Phase Ingredient Amount Aqueous phase 1 Water filling up to 100% (W1)Citric acid 0.6%   Glycerol 5% Butylene glycol 5% Galacto arabinane0.3%   Parabens in phenoxyethanol 0.8%   Oily phase 1 (O1)Polyglyceryl-3-methylglucose 2.5%   distearate Hydrogenatedpolyisobutene 3% Vegetable oil 4% Dicapryryl ether 3% Behenyl alcohol 2%Dimethicone 0.5%   Oily phase 2 (O2) Maize phosphates 1% Dimethicone0.5%   Plant extract (A) Plant cell extract of Example 4 2%

Instruction for Preparation:

Aqueous phase is mixed and heated to 80 centigrade. Oily phase 1 isheated to 80 centigrade, and oily phase 2 is added. The combined aqueousand oily phases are mixed and homogenized. The mixture is chilled to 30centigrade, and phase A (plant extract) is added, and the blend is mixedagain.

Example 9 In-Vitro-Test on Stem Cells

The test was carried out on stem cells originating from the umbilicalcord. The cells were grown in a complex medium containing 10 percent offetal calf serum. The supernatant without cell debris was used for thetest. Previous to the test, the extract was sterilized by filtration.

The addition of 0.1 percent of the extract resulted in a increase of thecell count of about 44 percent (FIGS. 1 and 3).

Also, in a subsequent MTS-assay on addition of 0.1 percent of theextract an increase of the proliferation capability of the cells of 20percent could be verified (FIG. 2).

In addition to said growth and proliferation studies umbilical cordcells together with a liposomal extract of dedifferentiated cells ofApples of the cultivar Uttwiler Spaetlauber were tested for the effectsof UV radiation. Application of 0.1 percent of extract resulted in areduction of the proliferation capability of about 7 percent, whereasthe control preparation showed a loss of proliferation capability of 42percent (FIG. 4).

Example 10 Ex-Vivo-Test on Isolated Hair Follicles

The epithelium of the hair root bulges into a suprabasal bulge which isthe niche of the ceratinozyte stem cells. They consist of clonalsubpopulations which regenerate skin and hair follicles. Thus, isolatedhair follicles are a suitable model for analyzing the life expectancy ofstem cells.

Hair follicles were isolated from skin material originating from anesthetic surgery. Then they were placed in a nutrient solution wherethey lived and started growing. In this manner, hair follicles could bekept alive for about 14 days. Thereafter, the cells begin to die off,and the newly formed hair begins to shrink. a control assay of 12follicles was incubated in the nutrient solution only, Whereas a secondseries was incubated in a nutrient solution containing 0.2 percent of aliposomal extract of dedifferentiated cells of apples of the cultivarUttwiler Spaetlauber. On the 16th, 18th and 20th day the length of thehair follicles was measured.

The ex vivo test showed that, as excepted, the follicles of the controlassay had lost about 6 percent of its length already on the 16th day. Asimilar shrinking could be asserted on the 18th day. Then, on the 20thday a considerable dying of 52 percent was measurable. The folliclestreated with the extract remained longer in the growth phase. On the16th day, an increase in length of 8 percent could still be measured.Not until the 18th day a slight shrinking arose. The dying on the 20thday was clearly lesser than in the control assay.

In detail, the following variations in length were detected. They aregraphically represented in FIG. 5.

Control Extract 16th day −5.7%  7.8% 18th day −5.3% −4.8%  20th day −52% −35%

Thus, Example 10 shows that a liposomal extract of dedifferentiatedcells of apples of the cultivar Uttwiler Spaetlauber is able to prolongthe expectancy of life of cerationocyte stem cells.

Dermatologic Tests Test 1 Anti-Wrinkle Effect of PhytoCellTec™ MalusDomestica

The following dermatological test was carried out by Dr. H. P. Nissen,of Derma Consult GmbH, D-53347 Alfter, Germany. PhytoCellTec™ MalusDomestica is the Applicant's Trade Mark for products prepared inaccordance with the present invention.

Test Product

-   -   Cream containing 2.0% of PhytoCellTec™ Malus Domestica

Test Area

-   -   Crow's feet area

Volunteers

-   -   Number of individuals: 20    -   Age: 37 to 64 years    -   Sex: female

Application

-   -   Duration: 28 days    -   Frequency: twice daily

Test Parameter

-   -   Wrinkle depth by means of an apparatus PRIMOS® 5.5 of        GFMesstechnik GmbH, D-14513 Teltow, Germany

Study Design

-   -   Day 0

Determination of the Test Parameter in the Test Areas; First Applicationof the Test Product

-   -   Day 1 to 13    -   Application of the test product twice a day    -   Day 14    -   Determination of the test parameter 8 to 12 hours after the last        daily test product application    -   Day 15 to 27:    -   Application of the test product twice a day    -   Day 28:    -   Determination of test parameter 8 to 12 hours after the last        daily test product application

Results

Two daily applications of the test cream containing 2% of PhytoCellTec™Malus Domestica over 28 days resulted in a significant decrease inwrinkle depth in all of the volunteers tested. These results arerepresented in FIG. 6.

Test 2 Effect of PhytoCellTec™ Malus Domestica on Stressed Skin

The following dermatological test was carried out by F. Juchaux, ofBIOalternatives, F-86160, France. PhytoCellTec™ Malus Domestica is theApplicant's Trade Mark for products prepared in accordance with thepresent invention.

Introduction

In normal skin the tumor suppressor gene p53 is upregulated by severaltypes of stress, e.g. DNA damage (induced by UV radiation, IR radiation,or chemical agents, such as hydrogen peroxide), oxidative stress, orosmotic shock. The protein p53 plays an important role in the cell cycleas transcription regulator. In old skin this gene is no more upregulatedbut rather down regulated by stress.

Test Product

2.0% of PhytoCellTec™ Malus Domestica

Cells

Normal human dermal fibroblasts (NHDF) used at 10th passage

Procedure

Fibroblasts were stressed for 2 hours with culture medium containing 600μmole of H₂O₂. For recovery, the cells were incubated for 72 hours witha medium containing, or not containing (control), 2% of PhytoCellTec™Malus Domestica. After the incubation time, mRNA was extracted andtranscribed into ³³P-labeled cDNA via reverse-transcription. Theselabeled cDNA targets were hybridized to an “old skin” specific minichip.This minichip contained about 150 genes specific for skin aging. Thecontent of labeled genes on the minichip was measured.

Results

In H₂O₂-stressed fibroblasts, p53 was downregulated. H₂O₂-stressed cellstreated with 2% PhytoCellTec™ Malus Domestica showed an upregulation ofp53. These results are represented in FIG. 7.

1. A cosmetic preparation for the treatment of skin stem cells,comprising: a dermatologically suitable carrier suitable for exteriorapplication to the human body; and an active component blended with saidcarrier, said component including an extract obtained from high pressurehomogenization of cultivated dedifferentiated plant cells in thepresence of finely dispersed liposomes encapsulating and stabilizingsaid extract.
 2. The cosmetic preparation of claim 1, wherein saiddedifferentiated plant cells originate from plants of the family ofRosaceae.
 3. The cosmetic preparation as set forth in claim 2, whereinsaid dedifferentiated plant cells originate from plants of thesub-family of Maloidae.
 4. The cosmetic preparation as set forth inclaim 3, wherein said dedifferentiated plant cells originate from plantsof Malus domestica cultivar Uttwiler Spaetlauber.
 5. The cosmeticpreparation as set forth in claim 1, comprising 0.01 to 90 percent byweight of said active component relative to said carrier.
 6. Thecosmetic preparation as set forth in claim 5, comprising 0.1 to 10percent by weight of said active component relative to said carrier. 7.A cosmetic preparation as set forth in claim 5, wherein said activecomponent is provided in an effective amount to promote proliferation,protection and vitalization of said stem cells.
 8. A cosmeticpreparation as set forth in claim 5, wherein said active component isprovided in an effective amount to protect hair follicle stem cells. 9.The cosmetic preparation of claim 1, wherein said finely dispersedliposomes are of size of about 50 nanometer.
 10. The cosmeticpreparation of claim 1, wherein said dedifferentiated plant cells arehomogenized at a pressure of about 1200 bar.
 11. A cosmetic preparationas set forth in claim 1, wherein said cosmetic preparation furthercomprises pharmaceutically and cosmetically compatible preservativeagents and antioxidants.
 12. A cosmetic preparation as set forth inclaim 1, wherein said cosmetic preparation is provided in creams, soaps,lotions, gels or hair serum.
 13. A method of preparing adedifferentiated vegetable stem cell suspension suitable for use in acosmetic preparation as set forth in one of the preceding claims,comprising the following procedure: decomposition of the plant cells byhigh pressure homogenization; and extraction and stabilization of theingredients by means of liposomes; wherein both parts of the method aresimultaneously carried out in a single step.